IE72525B1 - Method of preparing diacetyl rhein - Google Patents

Abstract

The present invention provides a process for treating arthritis utilizing substantially pure diacetylrhein having an aloe-emodin content of less than 20 ppm.

Description

Method of preparing dlacetyl rhein The invention relates to a method for the preparation of diacetylrhein in pharmaceutically usable purity having a residual content of unwanted aloe-emodin derivatives of 5 less than 20 ppm in total, the diacetylrhein obtainable by this method and a pharmaceutical preparation containing this compound.

Diacetylrhein of the formula: is a medicinally active substance which possesses antiarthritic, antiinflammatory, antipyretic and analgesic activity. For this reason diacetylrhein is used for the treatment of arthritic diseases, see for example DE-A 27 11 493 and US-A 4 244 968.

Diacetylrhein can be prepared, for example, by acetylation of barbaloin and oxidation with chromium trioxide of the peracetylated barbaloin obtained. Diacetylrhein can also be prepared by acetylation of rhein, which can be isolated, for example, from the senna drug (see DRUGS OF THE FUTURE, Vol.IV, N.E. 1979, pages 445 - 447).

Diacetylrhein obtained by this method contains as unwanted accompanying substances aloe-emodin derivatives, which arise as a result of an incomplete oxidation by chromium trioxide or are coextracted during the extraction of the 25 senna drug. These accompanying substances are contained in - 2 relatively small quantities and can therefore be separated only with great difficulty by means of classical purification procedures. Moreover, in the first method mentioned above, chromium residues accumulate which have to be removed in a suitable manner.

The present invention is therefore based on the object of providing a method for obtaining diacetylrhein which is easy to carry out and produces high yields and whereby diacetylrhein is obtained in pharmaceutically usable purity having a residual content of unwanted aloe-emodin derivatives of less than 20 ppm in total.

This object is fulfilled by the method according to the invention, which is characterised in that A) rhein-9-anthron-8-glucoside containing aloe-emodin components (that is, aloe-emodin and/or derivatives thereof) is subjected to a liquid-liquid partition between a polar organic . solvent which is only partly miscible with water and an aqueous phase, B) the rhein-9-anthron-8-glucoside contained in the aqueous phase after the partition is oxidised to rhein-8-glucoside, C) the glucose radical in the 8-position of the rhein-9glucoside is split off in an acid medium and D) the rhein obtained is acetylated and the diacetylrhein is recovered.

The sennosides contained in the senna drug are an important source of rhein-9-anthron-8-glucoside. A preferred embodiment of the invention is therefore a method for the preparation of diacetylrhein which is substantially free from aloe-emodin derivatives, whereby A) a mixture of sennosides is reduced to the corresponding rhein-9-anthron-8-glucoside compounds and aloe-emodin-9-anthron-8-glucoside compounds, B) a liquid-liquid partition of the compounds obtained is carried out between a polar organic solvent which is only partly miscible with water and an aqueous phase, C) the rhein-9-anthron-8-glucoside compounds contained in the aqueous phase after the partition are oxidised to the corresponding anthraquinone compound, D) the glucose radical in the 8-position of the anthraquinone compound is split off in an acid medium and E) the 1,8-dihydroxyanthraquinone compound obtained is acetylated and the diacetylrhein is recovered.

The individual steps in the method are explained below.

Reduction of the sennosides The mixture of sennosides used as starring material can be isolated, for example, from the senna drug.

The senna drug consists of the dried leaves and fruits of the senna plant, for example, the Indian senna (Cassia angustifolia) and the Egyptian senna (Cassia acutifolia). The senna drug contains dianthrone glucosides of rhein and aloe-emodin. The most important are the sennosides A, B, Al, C, D and DI. The sennosides correspond to the formula: CSH11°5~° ° 0R· In the sennosides A, B and Al, R represents COOH and in the sennosides C, D and Dl, R represents CH2OH. The sennosides A, B and Al and C, D and Dl respectively are stereoisomers and differ from one another in the configuration on the C atoms 10 and 10'.

The recovery of the sennosides from the senna drug is described, for example, in DE-A 32 00 131; here reference is made to the whole specification. According to this, the senna drug is first of all extracted with aqueous methanol. The concentrate remaining after complete removal of the methanol contains the sennosides in the form of the potassium salt. This concentrate is suitable for use as starting material for the method according to the invention. The concentrate can in addition be purified by liquid extraction with alcohols or ketones which are partly soluble in water, for example, butanol-2, 2-butanone (raffinate). The raffinate is acidified to a pH value of about 1.5 to 2.0 and the sennosides are crystallised out by seeding. The mixture of raw sennosides obtained can likewise be used as starting material for the method according to the invention. The mixture of raw sennosides may also be further recrystallised if desired.

Alternatively, the concentrate together with an added alcohol or ketone partly soluble in water, in particular butanol-2, can be used as starting material.

During the extraction of the senna drug, the proportion of drug to extracting solvent is preferably from l : 4 to 1 : 15, in particular from 1 : 4 to 1 .- 10.

The extraction is carried out preferably in the presence of a buffer, for example, trisodium citrate, glycine, sodium bicarbonate or saccharose.

In the method according to the invention, these starting materials are subjected to a complete reduction to., the corresponding rhein-9-anthron-8-glucoside (R = COOH) and to the corresponding aloe-emodin-9-anthron-8-glucoside (R = CH2OH) of the formula: Reducing agents having a suitable reduction potential are, for example, tin(II) chloride, sulphur dioxide, alkali metal borohydrides and preferably alkali metal dithionites, in particular sodium dithionite.

To carry out the reduction, the starting material can be in aqueous solution or in suspension and the reducing agent can be added thereto in solid form or dissolved in water.

It is also possible to operate in a two-phase mixture by adding thereto a polar organic solvent which is partly miscible with water, in particular 2-butanol or acetone.

The- reduction can be performed at ambient temperature or at elevated temperatures. It is advisable to carry out the reduction at 40 to 60°C, in particular at 50 to 55°C. The operation is performed using a starting solution or suspension of sennosides having a weakly acid to weakly alkaline pH value, preferably from pH 7 to 9. If desired, the reduction can be carried out several times, in particular 2 to 10 times.

The 9-anthron-8-glucosides formed are precipitated out by the addition of an acid, for example, sulphuric acid, until the pH is 4 to 4.5. It is advisable that the temperature in 10 the course of this should be not more than 40°C. It is advisable to operate under nitrogen during the precipitation of the 9-anthron-8-glucosides and during the isolation thereof (for example, by filtration), in order to avoid an uncontrolled oxidation of these compounds.

It is essential that the reduction proceeds to completion. It is advisable, therefore, to use the reducing agent in large excess. In the case of sodium dithionite, generally 1 to 4 times the quantity by weight of sodium dithionite is used, referred to the content of sennosides in the starting 20 material. In addition the reducing agent is allowed to react for at least 2 hours, preferably for at least 3 hours. In general the reduction is not carried out for longer than 10 hours. It is preferable to carry out a further reduction under the aforesaid conditions.

Prior to being used in the subsequent step, the product obtained is preferably reprecipitated by bringing it into aqueous solution by the addition of a base (NaOH, KOH) until the pH value is about 6 to 7, extracting the aqueous solution with 2-butanol, acetone or 2-butanone and again precipitating out the product by the addition of an acid until the pH value is about 2 to 4.

Liquid-liquid partition In this step the aloe-emodin components, in particular aloe-emodin-9-anthron-8-glucoside, are removed. For this purpose a liquid-liquid partition of the product obtained is carried out in a polar organic solvent which is only partly miscible with water and an aqueous phase. Suitable polar organic solvents are C4 - C5 alkanols and Cx - C3 dialkyl ketones, such as acetone, 1-butanol, 2-butanol and 2-butanone. Preferably 2-butanol or acetone are used.

A reducing agent is preferably added to the aqueous phase in order co impart to the aqueous phase a redox potential of -210 mV, or more negative, throughout the liquid-liquid partition. It is appropriate to use Che same reducing agent as in Step A. When an alkali metal dithionite is used as reducing agent, a 2 to 4 wt.% solution at a pH value of from 7 to 11 is generally sufficient to maintain the abovementioned conditions regarding the potential.

The volume ratio of aqueous phase (heavy phase) to organic phase (light phase) is generally within the range of from 1 : 5 to 1 : 40.

The liquid-liquid extraction is preferably carried out in countercurrent. Here the mixture of anthrone compounds is introduced in the form of the solution obtained after the reduction or, if the anthrone compounds have been isolated, in the form of a 3 to 15 wt.% solution.

After the partition the desired rhein-9-anthron-8-glucoside is present in the aqueous phase. It is precipitated out by adding an acid until the pH value is about 2 to 4, and then recovered in the conventional manner.

Oxidation of rhein-9-anthron-8-glucoside The rhein-9-anthron-8-glucoside obtained is now oxidised to R = COOH, CH2OH Oxidising agents suitable for this purpose are, for example, oxygen, peroxide compounds (hydrogen peroxide), compounds of manganese, chromium or iro,n in the high oxidation states. Preferably an iron(III) salt is used, in particular iron(III) sulphate. It is advisable to operate at elevated temperature, but to maintain it below 60°C. The formation of unwanted and undefinable oxidation products is thereby avoided. After completion of oxidation the rhein-8glucoside formed is isolated in the conventional manner.

Splitting off of the glucose radical The glucose radical in the 8-position is split off in acid solution. It is advisable to operate at about 85 to 95°C. The product obtained is isolated in the conventional manner.

The conversion of sennosides, after acid hydrolysis, directly into rhein by reaction with iron(TIT) chloride is known, see for example DE-A 27 11 493. But the yield in this case is only about 10% and in addition the rhein formed is difficult to separate.

In the method according to the invention the reductive splitting of the sennosides, the oxidation of the anthrone compounds formed to the corresponding anthraquinone compounds and the splitting off of the glucose radical in the 8-position of the anthraquinone compounds are each carried out in separate steps. Following the reductive splitting, all compounds which may lead subsequently to the formation of aloe-emodin or derivatives thereof are removed quantitatively by liquid-liquid partition. Furthermore, it is possible to carry out the oxidation at mild temperatures, so that the formation of unwanted and undefinable oxidation products is avoided. Moreover, in this reaction procedure the iron salt used can be recovered virtually quantitatively and used again after reoxidation. The separation of the oxidation step and the hydrolysis step, owing to the greater solubility in water of the anthrone glucosides as compared with the aglycones in question, permits the oxidation to be carried out gently at room temperature or below 60°C, the otherwise unavoidable formation of undefinable secondary products thereby being avoided.

Acetylation of the 1,8-dihydroxyanthracfuinone compound The acetylation of the 1,8-dihydroxyanthraquinone compound obtained is carried out in a conventional manner. For example, acetylation can be carried out using acetic anhydride in the presence of sodium acetate, as described in Arch. Pharm. 241, 607 (1903) . The acetylation may however also be carried out by other methods known to the person skilled in the art, for example, by reaction with acetyl chloride et cetera.

The diacetylrhein obtained in this way is substantially free from aloe-emodin and derivatives thereof. The content of these impurities is still about 50 ppm (determined by the analytical methods described in the Examples). The content of these impurities can be further lowered if the diacetylrhein obtained is recrystallised in the following manner.

The diacetylrhein is converted into an alkali metal salt by treatment with a suitable base. An example of a suitable base is an alkali metal acetate, preferably potassium acetate. Preferably equimolar quantities of the base and of an aqueous Cx - C3 alcohol, for example, 80% to 90% ethanol, are used as the reaction medium. The alkali metal salt of the diacetylrhein is allowed to crystallise out in the cold, then taken up in an aqueous Cx - C3 alcohol and precipitated out by the addition of an acid until the pH value is about 3. The precipitated diacetylrhein is then isolated and worked up in the conventional manner.

The product thus obtained contains less than 20 ppm of the above-mentioned impurities. Furthermore, the product is present in the form of needle-shaped crystals, which are particularly suitable for pharmaceutical formulation.

The product can be dried in the conventional manner. It is advisable to carry out the drying first of all in a vacuum at a relatively low temperature, for example, not more than 40°C, until the water content of the product has fallen to about 3% or less. The temperature can subsequently be elevated to 70 to ll0°C.

The invention also relates to the substantially pure diacetylrhein obtainable according to the invention and to a pharmaceutical preparation containing this compound. The fields of application, the dosage to be administered and suitable forms of administration are known,· see, for example, US-A 4 244 968, 4 346 103, 4 950 687, DE-A 27 11 493 and Drugs Exptl. Clin. Res. 6 (1) 53 to 64 (1980) .

The invention is illustrated by the following Examples.

Examole l Isolation of the mixture of sennosides used as starting material: In each case 40 kg of senna drug (sennoside content about 1.5%) are introduced into two percolators connected in series, having a volume of 250 1, and covered with a perforated steel plate. The solvent used for the extraction is 70% methanol, which is passed to the drug in the first percolator. The solution formed in the first percolator is passed to the drug contained in the second percolator. In the course of this the solvent is allowed to flow freely through the first percolator. 160 1 of solvent in total is used for the extraction of 40 kg of senna drug. After this volume of 70% methanol has been passed through the two percolators and the corresponding amount of percolate has been collected, the discharge tube of the percolator is connected to a postpercolate container and a further 60 1 of 70% methanol is additionally passed through the percolators. Afterwards the free solvent remaining from the first percolator is passed into the upper part of the second percolator and the postpercolate is collected until it amounts to 120 1 in total. The first percolator is then emptied, again filled with 40 kg of senna drug and the post-percolate is pumped onto the drug, with 120 1 of post-percolate being sufficient to cover the drug in the percolator. The temperature of the solution is then adjusted to +30°C.

This percolate is combined with that previously extracted and the extraction is carried out as described above.

For each 40 kg of drug 160 ml of percolate is collected, from which the methanol is removed in a rotary vacuum evaporator equipped with a packed column. About 30 1 of bottom product is obtained. This concentrate is extracted with the identical volume of water-saturated butanol-2.

Step A: Reduction of the sennosides to rhein-9-anthron-8-glucosides 1.0 1 of the extracted concentrate is brought to a pH value of 7.5 using 48% sodium hydroxide solution. It is heated to 60°C and 90 g of sodium dithionite in solid form is added to the solution, with stirring, over a period of half an hour. After the addition has been concluded, the solution is stirred for a further hour. Subsequently concentrated sulphuric acid is added, with stirring, until a pH value of 2 is attained. The solution is cooled to ambient temperature over a period of two hours and the crystalline deposit precipitated out is filtered off and washed with water containing sulphur dioxide.

If desired, the crude rhein-9-anthron-8-glucoside is reprecipitated. The filter cake is dissolved, while still moist, in a mixture of 15 parts by volume of 2-butanol and 85 parts by volume of water, which mixture contains 0.5 wt.% of sodium pyrosulphite, so that a 10% solution (w/v) is obtained by the addition of 48% of sodium hydroxide solution until the pH value is 7. The solution is acidified to a pH value of 2.8 or less using concentrated hydrochloric acid and allowed to stand for 2 hours. The precipitate deposited is filtered off, washed with water containing sulphur dioxide or sodium pyrosulphite, and dried.

Yield: 90% A further reduction (post-reduction) of the product thus obtained is carried out in the following manner: 3.0 g of the crude dried rhein-9-anthron-8-glucoside or the corresponding quantity of the moist product is dissolved in 15 ml of water together with 1.4 g of sodium dithionite and 2.3 ml of 5N NaOH. The solution is then made up to a volume of 24 ml with water and heated for 20 minutes at 55°C. Thereafter a further 1.5 g of sodium dithionite is added to the solution and this is heated for 20 minutes at 55°C. 0.9 ml of 5N NaOH and 1.5 g of sodium dithionite are then added. After the solution has been heated for 20 minutes at 55°C, 0.9 ml of 5N NaOH is once again added. The solution obtained is introduced directly into the subsequent liquid-liquid extraction.

Step B: Separation of the aloe-emodin components The separation of the aloe-emodin components is carried out by liquid-liquid partition of the 9-anthron-8-glucosides in countercurrent by means of an apparatus consisting of 60 mixer-settler units (mixer-settler apparatus). A solution of 3.0 g of sodium dithionite in 3.5 ml of 5N NaOH and 96 ml of water is used as the aqueous, heavier phase. (Water-saturated) 2-butanol or acetone is used as the organic, lighter phase. The two phases are passed through the apparatus in such a way that the volume ratio of heavy phase to light phase is 1 : 10.

The mixture to be separated is supplied to the apparatus in the form of the freshly reduced solution or in the form of a solution of corresponding pH value and corresponding concentration, which solutions contain the 9-anthron-8glucosides obtained from Step A, namely, in such a manner that 30 parts by volume of the organic phase are used per one part by volume of the mixture to be separated.

The pH value of the solution containing the mixture is maintained at 9 to 9.5 by means of a glycine buffer. The buffer, consisting of 3 parts by volume of a 7.5% glycine solution and 1 part by volume of IN NaOH, is added in a quantity of 240 ml of buffer solution per 150 g of crude rhein-9-anthron-8-glucoside. The unwanted aloe-emodin compounds accumulate in the organic phase, whereas the rhein-9-anthron-8-glucoside remains in the aqueous phase. The aqueous phase is acidified to a pH value of 2.8 using sulphuric acid, the precipitate formed is filtered off, washed with water and acetone, and dried in the air at ambient temperature. In this way rhein-9-anthron-8glucoside containing 41 ppm of aloe-emodin components is obtained. The content of aloe-emodin components is determined as aloe-emodin, using a method which is described at the end of this patent application.

Yield: 97%, referred to rhein-9-anthron-8-glucoside.

Step C: Oxidation to rhein-8-glucoside The product from Step B (referred to the content of 3.0 kg of sennosides A, Ax and B) is suspended in a solution of 184 1 of demineralised water and 75.5 kg of iron(III) sulphate hydrate (22% Fe3+) . The suspension is heated to between 55 and 62°C and oxidised for 14 hours with the use of a rapidly flowing dispersant. If oxidation is complete, the rhein-8-glucoside formed is filtered off and washed with 50 1 of demineralised water which has been adjusted to pH 2 using sulphuric acid.

Step D: Hydrolysis to rhein The moist filtration residue from Step C is suspended in 200 kg of 20 wt.% of sulphuric acid and stirred for 8 hours at 88 to 92°C. The rhein formed is filtered off and can be dried in a 1 mbar vacuum for 4 8 hours at 40°C for storage, or else be used immediately in the moist condition for the acetylation in Step E.

Overall yield for the steps A to D: 79%, referred to the sennosides A, Ax and B used in Step A.

Step E: Acetylation to diacetylrhein 6.5 kg of rhein from Step D is suspended in 100 1 of acetic anhydride for 10 minutes and 2 kg of potassium acetate is added thereto. The reaction mixture is heated to 95°C with stirring, 0.65 kg of activated carbon is added and stirring is continued for half an hour at 90 to 95°C. The activated carbon is filtered off from the hot solution and 2.1 kg of 96 to 98 wt.% sulphuric acid is added to the filtrate. The reaction mixture is cooled to 20°C as rapidly as possible, with stirring. The resulting suspension is filtered. The residue is washed free of sulphate with demineralised water.

Yield: 83% Step F: Recrystallisation, drying, grinding 7.5 kg of diacetylrhein from Step E (referred to the dried substance) is suspended, with rapid stirring, in 375 1 of 90 vol.% ethanol. The suspension is heated to 70°C and then 3.75 kg of potassium acetate is added thereto. On cooling to 0 to 2°C, the pure potassium salt of diacetylrhein crystallises out from the clear solution formed in the meantime.

The potassium salt is filtered off and dissolved in 300 1 of 40 vol. % ethanol at 20 to 30°C, with 3 kg of potassium acetate being added. The clear solution is adjusted to a pH value of 3.0 by means of 10 wt.% sulphuric acid. The diacetylrhein crystallised out is filtered off and washed free of sulphate with demineralised water.

The product is first of all dried in a vacuum at 1 mbar and at 40°C within 24 hours. If the residual water content has fallen below 3%, the material is coarsely comminuted and further dried in a 1 mbar vacuum and at 70°C for 24 hours.

The product is then ground using a 0.5 mm strainer and, in order to remove solvent residues, is further dried in a 1 mbar vacuum and at 70°C.

Yield: 95% Example 2 The extraction of the senna drug and the reduction of the sennosides described in Example 1 are repeated. The postreduction is then carried out in the following manner: 14.0 g of saccharose, 4.5 g of sodium dithionite (85%) and 13.3 g of potassium acetate are dissolved in 133 ml of water and thereto are added 1.3 ml of 48% sodium hydroxide solution and 17.3 g of potassium carbonate. Subsequently 293 ml of acetone and 50 ml of water are added. The mixture is shaken in a separating funnel and the phases are separated, with 375 ml being obtained in the upper phase (acetone phase) and 130 ml in the lower phase. 1.4 ml of 48% sodium hydroxide solution and 10 g of crude rhein-9-anthrone-8-glucoside are dissolved in 98 ml of the lower phase. The solution is heated to 45 to 50°C and maintained at this temperature for 20 to 30 minutes. 1.0 ml of 48% sodium hydroxide solution and 3.4 g of sodium dithionite are then added and the solution is heated for a further 20 to 30 minutes at 45 to 50°C. Subsequently 1.0 ml of 48% sodium hydroxide solution and 3.4 g of sodium dithionite are once again added and the, solution is heated for 20 to 30 minutes at 45 to 50°C.

The separation of the aloe-emodin component is carried out by liquid-liquid partition of the reduced solution in countercurrent to the above-mentioned upper phase (acetone phase) . The raffinate phase running off, which contains the rhein-9-anthron-8-glucoside, is concentrated to 400 ml by evaporation and 20 ml of butanol-2 is added thereto. Hydrochloric acid or sulphuric acid is added until the pH value is 4.0 to 4.2. The precipitate formed is filtered off, washed with 40 ml of water and 30 ml of acetone and then dried. The subsequent oxidation is carried out as described in Example 1.

Example 3 Approximately 2 1 of butanol-2 is added to the concentrate obtained after the extraction of the senna drug. The reduction of the senna fruit concentrate/butanol-2 mixture is then carried out in 7 steps under nitrogen as protective gas. A precipitation of the crude rhein-9-anthron-8glucoside is carried out after reduction step I.

Reduction step I: 100 1 of senna fruit concentrate/butanol-2 mixture, containing about 4 kg of sennosides, is placed in a stirrer vessel and covered with nitrogen. With stirring, 6 1 of 20 wt.% sodium hydroxide solution and then 350 1 of watersaturated butanol-2 (for example, from Step II) are added in succession, followed by stirring for 15 minutes. The batch is heated to between 42 and 50°C and 7 kg of sodium dithionite is added. Stirring is continued for 45 minutes. The pH value is maintained at 7.5 to 8 using 20 wt.% sodium hydroxide solution. The reduction potential (against an Ag/AgCl electrode) is maintained at below -630 mV, if necessary by addition of sodium dithionite. After cooling the reaction to 30 to 35°C, precipitation is effected within 1.5 hours by adding 10 wt.% sulphuric acid until the pH is less than 4. The resulting suspension is stirred at a temperature of below 25°C for about 10 hours at a low stirring speed. The resulting precipitate is filtered off. The precipitate is suspended in 60 1 of 15 wt.% butanol-2, stirred for 30 minutes at 50 to 60°C and subsequently filtered. The residue is washed with 100 1 of demineralised water. The yield of crude rhein-9-anthron-8-glucoside, referred to the sennosides used, is above 82%.

Reduction step II 3.3 kg of crude rhein-9-anthron-8-glucoside from Step I is suspended in a mixture of 42 1 of demineralised water and 7.4 1 of butanol-2. The suspension is brought into solution using 2 1 of 20 wt.% sodium hydroxide solution and 9.9 kg of trisodium citrate and thereafter 3.3 kg of sodium dithionite and 350 1 of water-saturated butanol-2 (for example, from Step III) are added. The batch is heated to between 42 and 45°C. The pH value is maintained at 8.5 to 9 using 20 wt.% sodium hydroxide solution. The reduction potential (against an Ag/AgCl electrode) is maintained at below -750 mV, if necessary by addition of sodium dithionite.

After being left to stand for 30 minutes, the upper phase is removed and the lower phase is further processed in Step III .

Reduction step III The reduction/extraction process described in Step II is repeated with the lower phase from Step, II, with the addition of the following chemicals: 1.65 kg of sodium dithionite 0.8 1 of 20 wt.% sodium hydroxide solution 350 1 of water-saturated butanol-2 (for example, from Step IV) Reduction steps IV - VII The reduction/extraction process described in Step II is repeated with the lower phase from each of the preceding Steps, with the addition of the following chemicals: 0.825 kg of sodium dithionite 0.4 1 of 20 wt.% sodium hydroxide solution 350 1 of water-saturated butanol-2 (for example, from each of the subsequent steps - countercurrent principle) The lower phase separated off in Step VII is cooled to 30 to 35°C and the rhein-9-anthron-8-glucoside is precipitated out' as described in Step I. The resulting precipitate is filtered off and washed with 100 1 of demineralised water.

The precipitate is then covered with 10 1 of iron(III) sulphate solution (for the preparation, see Step B, Example 1).

The rhein-9-anthron-8-glucoside is then converted into the sennosides, as described in Example 1 or 2.

Pharmacological investigations The activity of diacetylrhein was determined in chronic inflammation models after oral administration. The following experimental models were used: cotton pellet granuloma in the rat and arthrosis in rabbits produced by intra-articular administration of vitamin A. a) Cotton pellet granuloma in the rat Young, sexually mature rats (n = 10) received 25 mg, mg or 100 mg of diacetylrhein/kg or 5 mg of indomethacin/kg or 100 mg of acetylsalicylic acid/kg daily for 5 days. A control group treated only with water was also conducted at the same time. The implantation of the pellets took place on the first day of treatment. Fresh and dry weights of the granuloma prepared at the end of the experiment exhibited a significant and clearly dose-dependent lowering compared with the control group. Here the action of 100 mg of diacetylrhein/kg corresponded approximately to the action of 5 mg of indomethacin or 100 mg of acetylsalicylic acid. The weights of the thymus and of the adrenal glands did not change during the treatment. b) Vitamin A arthrosis An arthrosis-like change in the joints was produced in two groups each of 10 rabbits (white New Zealander) bymeans of three intra-articular injections of 30,000 IU of vitamin A over the course of 9 days. 56 days later, 10 animals were treated with 3 mg of diacetylrhein/kg/day for 8 weeks. In comparison with the control group, the macroscopically and microscopically detectable changes in the joints in the treated group were significantly lowered.

The curative action of diacetylrhein was also compared with that of acetylsalicylic acid in each of 7 rabbits which, after 6 days of pretreatment with three times 10,000 IU of vitamin A and a 26-day interval without treatment, received over 8 weeks either 5 mg of diacetylrhein/kg/day (experimental group) or 15 mg of acetylsalicylic acid/kg/day (positive control group), or remained untreated (negative control group). In all three groups comparable motor disturbances, in the form of dragging of the hind legs, appeared 24 days after the final vitamin A injection. In the negative control group the clinical symptoms of a manifest arthrosis increased in the course of the following 8 weeks .

In the experimental group and the positive control group these symptoms improved significantly during the 8 weeks of treatment.

Chancres in the gastric mucous membrane Whereas the single administration of 400 mg of dia-cetylrhein/kg or of the solvent caused no erosions of the gastric mucous membrane in the rat, after the administration of Ibuprofen (200 mg/kg) or Indomethacin (20 mg/kg) there was found unmistakeable damage to the mucous membrane in the form of punctate (1 mm diameter) to large (3 mm diameter) erosions. The twice daily administration of 100 mg of diacetylrhein/kg over 3 days produced no damage to the mucous membrane, whereas the corresponding application of 10 mg of indomethacin certainly did so, the erosions in this case having a diameter of 1 to 3 mm.

Toxicology The acute toxicity LD50, depending on the species investigated (rat, mouse, cat), is 1.9 to 7.9 g/kg following oral administration. Here the rat proved to be the least sensitive. After parenteral administration (i.v., i.p.) the LD50 values in these species was between 119 and 339 mg/kg.

Clinical trials 1. The action of diacetylrhein was investigated in coxarthrosis and gonarthrosis in 95 (49/46) patients in a double-blind study against Naproxen and subsequent placebo aftertreatment. The dosage administered was 50 mg of diacetylrhein twice daily or 750 mg of Naproxen daily. The duration of treatment was 60 days after a 7-day wash-out phase. The subsequent placebo treatment extended over 60 days .

Test variables were the pain and mobility symptoms according to a scale of scores, functional restriction and compatibility.

In both treatment groups (diacetylrhein/Naproxen), a statistically significant improvement rate (P < 0.01 and P < 0.05, respectively) with regard to all test variables was ascertained in comparison with the initial values. After discontinuation of the treatment and subsequent administration of the placebo, however, on days 90 and 120 a statistically significant superiority (P < 0.01) with regard to the variables spontaneous pain and active and passive motor pain was shown for the diacetylrhein/placebo group as compared with the Naproxen/placebo group. This difference was also obtained at the 5% level for the variables night pain and pressure pain 30 days after discontinuation of diacetylrhein. 2. In an open running study with a control, the action of diacetylrhein on osteoarthrosis of the spine and of the knee was investigated in 70 patients (35/35). The dosage administered was 100 mg of diacetylrhein per day. The duration of treatment was 60 days and the duration of observation was 75 days. The test variables were limitation of pain and restriction of movement. The variables were determined according to a score system.

The control group comprised 35 patients, who received exclusively physiotherapeutic treatment. Physiotherapy was also carried out on the group treated with diacetylrhein.

With regard to all variables, the evaluation of the results showed a statistically significant superiority in the treated group as compared with the control group. Even after discontinuation of the treatment a sustained therapeutic effect (hang-over effect) could be ascertained for the diacetylrhein group. 3. ' The action of diacetylrhein on localised arthrosis in 20 patients was investigated in a single-blind, cross-over study against Naproxen. The patients were divided into two groups, with 50 mg of diacetylrhein initially being administered to the first group twice daily for 20 days. Subsequently there followed a three-day wash-out phase and a further treatment with 250 mg of Naproxen twice daily for a further 20 days. In the second group the reverse sequence was followed. The duration of treatment was 43 days in total. Test variables were pain, compression pain, passive motor pain, functional restriction and swelling, determined according to a score system.

The evaluation of the results showed a superiority of the treatment with diacetylrhein in comparison with the treatment with Naproxen. No significant side effects were observed, nor any changes in the clinical laboratory variables . 4. The action of diacetylrhein was investigated in 23 patients (12/11) with osteoarthrosis in a randomised double-blind study using the double dummy technique against Naproxen (compatibility study). The dosage administered was 50 mg of diacetylrhein twice daily and 250 mg of Naproxen three times daily. The duration of treatment was 4 weeks. The test variables were the results of oesophagogastroduodenoscopic investigations before and after therapy. Only patients found to have normal mucous membranes or having slight mucosal lesions (grade 1) were included in the study.

After 4 weeks the endoscopic findings showed grade 2 mucosal lesions in one case (10%) in the diacetylrhein group, whereas in the group treated with Naproxen 5 patients (50%) exhibited grades 2, 3 and 4 mucosal lesions. The uptake was found to be normal in all cases.

Analytical determination of aloe-emodin mg of diacetylrhein is dissolved in 25.3 ml of 0.5 M NaOH in a separating funnel and shaken for minutes. 74.6 ml of a solution containing 0.5 M glycine and 0.5 M NaCl is subsequently added. A pH of 9.5 is thereby obtained.

This solution is extracted three times with 25 ml of chloroform. The combined organic phases are extracted once with 10 ml of 0.5 M of a buffer having a pH 9.5 (glycine, i0 NaOH, NaCl) and once with 10 ml of 0.01, M sulphuric acid.

The solvent is removed from the organic phase and the residue is dissolved in 1 ml of methanol.

To prepare the standard solution, 2 mg of aloe-emodin is dissolved in 20 ml of N,N-dimethylacetamide and diluted with methanol to a concentration of 2 ^g/ml (corresponding to 40 ppm) .

The content of the solutions is analysed by HPLC. The linearity of the HPLC method was verified using aloe-emodin standard solution in the range of from 0.11 ^g/ml (corresponding to 2.2 ppm) to 53.6 ^g/ml (corresponding to 1072 ppm) . The determination of the content is carried out using a Merck HPLC column Lichrocart 250-4, packed with LiChrospher-100 RP-18, 5 μπι, at 40°C with a mobile phase consisting of 1% acetic acid in methanol (v/v), 1% acetic 25 acid in water (v/v) and acetonitrile in the ratio of 49:46:5.

Analytical determination of the product from Step B, namely rhein-9-anthron-8-glucoside having a content of aloe-emodin components of 41 ppm, determined as aloe-emodin The substance to be investigated is converted into rhein 5 and aloe-emodin by oxidation with iron(III) chloride and simultaneous hydrolysis with hydrochloric acid in a 2-phase mixture of aqueous solution and carbon tetrachloride. The rhein is converted into a salt so that it can be separated from aloe-emodin by liquid-liquid partition. The aloe10 emodin present in the organic phase is determined by HPLC.

Claims (14)

1. Method for obtaining diacetylrhein which is substantially free from aloe-emodin components, characterised in that A) rhein-9-anthron-8-glucoside containing aloeemodin components is subjected to a liquid-liquid partition between a polar organic solvent which is only partly miscible with water and an aqueous phase, B) the rhein-9-anthron-8-glucoside contained in the aqueous phase after the partition is oxidised to rhein-8-glucoside, C) the glucose radical in the 8-position of the rhein-9-glucoside is split off in an acid medium and D) the rhein obtained is acetylated and the diacetylrhein is recovered.

2. Method for the preparation of diacetylrhein which is substantially free from aloe-emodin components, characterised in that A) a mixture of sennosides is reduced to the corresponding rhein-9-anthron-8-glucoside compounds and aloe-emodin-9-anthron-8-glucoside compounds, B) a liquid-liquid partition of the compounds obtained is carried out between a polar organic solvent which is only partly miscible with water and an aqueous phase, C) the rhein-9-anthron-8-glucoside compounds contained in the aqueous phase after the partition is oxidised to the corresponding anthraquinone compound, D) the glucose radical in the 8-position of the anthraquinone compound is split off in an acid medium and E) the 1,8-dihydroxyanthraquinone compound obtained is acetylated and the diacetylrhein is recovered.

3. Method according to claim 2, characterised in that an alkali metal dithionite is used as reducing agent in Step A.

4. Method according to claim 3, characterised in that the procedure is carried out at a pH value of from 7 to 9 .

5. Method according to any one of claims 2 to 4, characterised in that the reduction is carried out several times.

6. Method according to any one of the preceding claims, characterised in that acetone or 2-butanone is used as polar organic solvent for the liquid-liquid partition.

7. Method according to any one of the preceding claims, characterised in that an aqueous phase the redox potential whereof is -210 mV, or more negative, is used for the liquid-liquid partition.

8. Method according to any one of the preceding claims, - characterised in that the liquid-liquid partition is carried out in countercurrent. - 29

9. Method according to any one of the preceding claims, characterised in that an iron(III) salt, preferably iron(III) sulphate, is used as oxidising agent.

10. Method according to claim 2, characterised in that 5 the mixture of sennosides is obtainable by extraction of the senna drug with aqueous methanol, preferably in the presence of a buffer.

11. Method according to any one of the preceding claims, characterised in that the diacetylrhein obtained is 10 recrystallised by converting the said diacetylrhein into an alkali metal salt, by taking this up in an aqueous C x - .C 3 alcohol and again precipitating out the. diacetylrhein through the addition of an acid.

12. a method according to claim 1 for obtaining diacetyl15 rhein which is substantially free from aloe-emodin components, substantially as hereinbefore described and exemplified.

13. Diacetylrhein which is substantially free from aloeemodin components, whenever obtained by a method 20 claimed in a preceding claim.

14. A method according to claim 2 for the preparation of diacetylrhein which is substantially free from aloeemodin components, substantially as hereinbefore described and exemplified.